Within nucleic acid and genetic material technologies, it is often necessary to determine whether a gene, a part of a gene, or a nucleotide sequence is present in a living organism, a cellular extract of this organism, or a biological sample. Since any gene or part of a gene is characterized by a specific sequence of nucleotide bases, it is only necessary to search directly for the presence of all or part of said specific sequence in a sample containing a mixture of polynucleotides.
There is enormous interest in this search for specific polynucleotide sequences, particularly in detection of pathogenic organisms, determination of the presence of alleles, detection of the presence of lesions in a host genome, or detection of the presence of a particular RNA or modification of a cell host. Genetic diseases such as Huntington's disease, Duchenne's disease, phenylketonuria, and beta thalassemia can thus be diagnosed by analyzing nucleic acids from the individual. Also it is possible to diagnose or identify viruses, viroids, bacteria, fungi, protozoans, or any other form of plant or animal life by tests employing nucleic probes.
Once the specific sequence of an organism or a disease is known, the nucleic acids should be extracted from a sample and a determination should be made as to whether this sequence is present.
Various methods of nucleic acid detection have been described in the literature. These methods are based on the properties of purine-pyrimidine pairing of complementary nucleic acid strands in DNA-DNA, DNA-RNA, and RNA-RNA duplexes. This pairing process is effected by establishing hydrogen bonds between the adenine-thymine (A-T) and guanine-cytosine (G-C) bases of double-stranded DNA; adenine-uracil (A-U) base pairs can also form by hydrogen bonding in DNA-RNA or RNA-RNA duplexes. The pairing of nucleic acid strands for determining the presence or absence of a given nucleic acid molecule is commonly called “nucleic acid hybridization” or simply “hybridization”.
The most direct method for detecting the presence of a target sequence in a nucleic acid sample is to obtain a “probe” whose sequence is sufficiently complementary to part of the target nucleic acid to hybridize therewith. A pre-synthesised probe can be applied in a sample containing nucleic acids. If the target sequence is present, the probe will form a hybridization product with the target. In the absence of a target sequence, no hybridization product will form. Probe hybridization may be detected by numerous methods known in the art. Commonly the probe may be conjugated to a detectable marker. Fluorescent or enzymatic-markers form the basis of molecular beacons, Taqman and other cleavable probes in homogeneous systems. Alternatively the probe may be used to capture amplified material or labelled such that the amplicon is detected after separating a probe hybridized to the amplicon from non-hybridized material.
The main difficulty in this approach, however, is that it is not directly applicable to cases where the number of copies of the target sequence present in a sample is small, less than approximately 107 copies. Under these conditions it is difficult to distinguish specific attachment of a probe to its target sequence from non-specific attachment of the probe to a sequence different from the target sequence. One of the solutions to this problem consists of augmenting the detection signal by a preliminary technique designed to specifically and considerably increase the number of copies of a target nucleic acid fragment if it is present in the sample. A technique of this type is currently called an amplification technique.
The articles by Lewis (1992, Genetic Engineering News 12: 1-9) and Abramson and Myers (1993, Curr. Opin. Biotechnol. 4: 41-47) are good general surveys of amplification techniques. The techniques are based mainly on either those that require multiple cycles during the amplification process or those that are performed at a single temperature. Cycling techniques are exemplified by methods requiring thermo-cycling and the most widely used of this class of technology is PCR (polymerase chain reaction, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; European Patent No. 0 201 184) which enables the amplification of a region of interest from a DNA or RNA. The method usually consists of three steps:
(i) dissociating (denaturing) a double-stranded DNA into single-stranded DNAs;
(ii) annealing a primer oligonucleotide to the single-stranded DNA; and
(iii) synthesizing (extending) a complementary strand from the primer in order to copy a region of a DNA of interest.
After this process is completed the system is heated which separates the complementary strands and the process is repeated. Typically 20-40 cycles are performed to amplify genomic DNA to the extent that it can be Her analysed.
Variants of this process include repair chain reaction (RCR) (WO 90/01069) and reverse transcription-PCR (RT-PCR) (Trends in Biotechnology, 10:146-152 (1992)). Alternatively, in a reaction designated as “the shuttle PCR” (Tanpakushitsu Kakusan Kouso, Bessatsu, Protein, Nucleic Acid and Enzyme, Supplement, 41(5): 425-428 (1996)), two of the three steps, that is, the step of annealing the primer and the step of extending are carried out at the same temperature. The ligase chain reaction is an additional technique requiring multiple thermocycles and essentially copies the probe supplied to the system without amplifying target DNA.
A second class of amplification techniques, known as isothermal techniques, are those that are performed at a single temperature or where the major aspect of the amplification process is performed at a single temperature. In the sense that these reactions are performed under isothermal conditions they are more closely related to the subject of the current invention. In common with PCR, the isothermal techniques also rely on the ability of a polymerase to copy the template strand being amplified to form a bound duplex. In the multi-step PCR process the product of the reaction is heated to separate the two strands such that a further primer can bind to the template repeating the process. Conversely, the isothermal techniques rely on a strand displacing polymerase in order to separate/displace the two strands of the duplex and re-copy the template. This well-known property has been the subject of numerous scientific articles (see for example Y. Masamute and C. C. Richardson, 1971, J. Biol. Chem. 246, 2692-2701; R. L. Lechner et al., 1983, J. Biol. Chem. 258, 11174-11184; or R. C. Lundquist and B. M. Olivera, 1982, Cell 31, 53-60). The key feature that differentiates the isothermal techniques is the method that is applied in order to initiate the reiterative process.
Broadly isothermal techniques can be subdivided into those methods that rely on the replacement of a primer to initiate the reiterative template copying and those that rely on continued re-use or de novo synthesis of a single primer molecule.
One known process involving primer re-use is strand displacement amplification (SDA) (G. T. Walker, U.S. Pat. No. 5,455,166), in which a target nucleic acid sequence (and a complementary strand thereof) in a sample is further amplified after initial primer extension by displacement of the synthesised product by a strand displacing DNA polymerase. The strand displacing polymerase gains access to the template strand of the DNA at a site where the primer has been nicked by a restriction endonuclease. The method requires four primers, two of which should be designed to contain a recognition site for the restriction endonuclease.
Where amplification processes rely on primer replacement then the primer which is being replaced must be removed. Numerous methods have been described for primer removal during amplification. Whereas one method relies on the action of a DNA helicase to separate the extended primer from the template (US20040058378A1: Helicase dependent amplification of nucleic acids), other methods rely on the destruction of the primer by a nuclease. Clearly where a nuclease is utilised for primer destruction, the template being amplified must be protected from the action of the nuclease or the nuclease must be directed to specific elements unique to the primer itself.
European Patent No. 0 500 224 describes an exonuclease-mediated strand displacement amplification method which relies on an double strand specific 5′-3′ exonuclease that cleaves the extended primer. In this example a modified nucleotide triphosphate must be used within the reaction such that the copied template is resistant to digestion. The consequence of this schema is a non-exponential process.
Numerous primer replacement techniques rely on RNA/DNA composite primers wherein the primer may be removed by the application of the RNA/DNA hybrid specific enzyme RNAse H as exemplified by N. Kurn (WO0120035A2). This technology explicitly describes a linear amplification that does not include any reverse copying of the product strand and is therefore clearly distinguished from the current application. The isothermal amplification method as described by P. Cleuziat in U.S. Pat. No. 5,824,517 is a DNA amplification method that uses four primers at least two of which are chimeric, being composed of a 5′ RNA and a 3′ DNA element. The technique relies upon the four primers being in an overlapping, nested configuration to produce exponential amplification. An RNAse destroys the 5′ RNA element of the chimeric inner primers. This exposes the template strand such that the partially destroyed, extended inner primers are displaced by binding and then extension of the outer primers.
WO 99/49081 describes a method of amplifying DNA in which a duplex specific 5′-3′ exonuclease is used to partially digest a primer once it has bound to the template and extended. Thus, after initial binding and extension of a primer, the 5′ portion of the primer is destroyed by the 5′-3′ exonuclease, which allows a second, shorter primer to bind the exposed single-stranded template DNA. This process allows for exponential expansion of the template population but has an absolute requirement for the use of four primers in an overlapping nested configuration because a shorter primer (similar to the 5′ (digested) end of the original primer) is needed such that it wholly binds to the exposed template in order that the strand displacing polymerase may extend from the new primer and displace the initially formed product. Accordingly this technique makes use of two ‘full length’ primers (forward and reverse) and two shorter primers (forward and reverse) having sequence homology with the partially digestible regions of the ‘full length’ primers.
It is an object of the present invention to provide an alternative isothermal nucleic acid amplification technique.
It is a further object of the present invention to provide an exponential nucleic acid amplification technique.
It is a further object of the present invention to provide a nucleic acid amplification technique which requires only two primers.